Cancer-cell-specific cell proliferation inhibitors

ABSTRACT

The present inventors discovered that although suppressing expression of the RecQ1 gene, a RecQ helicase family gene, shows suppressive effects on cell proliferation in cancer cells, such effects are not observed in human TIG3 cells (a normal diploid fibroblast cell line), which are normal cells. Hence, the present inventors discovered that siRNAs against RecQ1 gene have cancer cell-specific cell proliferation-suppressing effects that are mediated by suppression of the expression of said gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

National Stage filing of PCT/JP2005/021099, International filing date ofNov. 17, 2005, which application claims priority to Japanese ApplicationNo. 2004-336742, filed Nov. 19, 2004.

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 390081_(—)403USPC_SEQUENCE_LISTING.txt. The textfile is 30 KB, was created on Aug. 4, 2011, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to compounds that suppress expression ofRecQ1 genes, and particularly relates to cell proliferation inhibitorscomprising siRNAs that exhibit the effect of suppressing expression ofthese genes.

BACKGROUND ART

Genes belonging to the RecQ DNA helicase family are widely present inorganisms ranging from prokaryotes such as Escherichia coli (E. coli) tohigher eukaryotes including humans. Conserved in the evolution process,these genes diversified along with the multicellularization oforganisms. The E. coli RecQ gene was the first of the RecQ family genesto be discovered. This gene was identified as a gene participating inzygotic recombination and in the RecF pathway for UV damage repair (seeNon-Patent Document 1). The E. coli RecQ gene has been revealed to havethe function of suppressing incorrect recombinations (see Non-PatentDocument 2). The budding yeast SGS1 gene and the fission yeast Rqh1 geneare the only known RecQ homologues in these yeasts. Both of these genesmainly suppress recombination and play important roles in genomestabilization (see Non-Patent Documents 3 and 4). Higher eukaryotescarry a number of RecQ homologues. In humans, there are five types ofgenes known to belong to the RecQ family: the RecQL1 (see Non-PatentDocument 6), BLM, WRN, RTS, and RecQL5 genes. Of these five, the RTSgene (see Non-Patent Document 5 and Patent Documents 1 and 2) and theRecQL5 gene (see Non-Patent Document 5 and Patent Document 3) wereidentified by the present inventors. The BLM, WRN, and RTS genesrespectively cause Bloom's syndrome (see Non-Patent Document 7),Werner's syndrome (see Non-Patent Document 8), and Rothmund-Thomsonsyndrome (see Non-Patent Document 9). These genes all play importantroles in genome stabilization in cells.

In fibroblast cells and lymphocytic cell lines derived from patientswith Werner's syndrome, chromosomal translocation and deletion, whichare indexes for genome instability, have been reported to occur withhigh frequency (see Non-Patent Document 10). Chromosomal breakage andsister chromatid exchange (SCE) are frequently detected in cells derivedfrom patients with Bloom's syndrome (see Non-Patent Document 11).Trisomies of human chromosome 2 and 8 are frequently found inlymphocytes derived from patients with Rothmund-Thomson syndrome (seeNon-Patent Document 12). These findings suggest that the WRN helicase,BLM helicase, and RTS helicase encoded by the various causative genes ofthese three genetic diseases play important roles in genomestabilization in cells.

Telomere length abnormalities are seen in lymphocytic cell lines derivedfrom patients with Werner's syndrome as compared to cell lines derivedfrom normal healthy subjects (see Non-Patent Document 13). In addition,cell immortalization was not observed in lymphocytic cell lines derivedfrom patients with Werner's syndrome, although about 15% of cell linesderived from normal healthy subjects were immortalized after passaging(see Non-Patent Document 14). This finding indicates that WRN helicasecontributes to telomere structure maintenance, and is thus essential forthe immortalization (canceration) of lymphocytic cell lines.

It has been suggested that WRN helicase is associated with homologousrecombination-mediated repair, because the helicase forms foci in thenucleus in response to DNA-damaging agents, and these foci areco-localized with the single-stranded DNA-binding protein RPA (which isa WRN-binding protein) and with the recombination repair factor RAD51(see Non-Patent Document 15). In addition, WRN helicase has been knownto bind to the DNA-dependent protein kinase complex (DNA-PK) and to flapendonuclease 1 (FEN-1). By binding to DNA-PK, WRN helicase plays animportant role in the processing of terminals generated by DNA doublestrand breaks, which are repaired in the pathway of non-homologous endjoining (see Non-Patent Document 16). WRN helicase is believed toactivate FEN-1 by binding to it, and to provide a site for precisereconstruction of the replication fork through homologous recombinationby processing Okazaki fragments (see Non-Patent Document 17). The abovefindings suggest that WRN helicase plays an important role in DNA repairduring DNA replication.

BLM helicase is localized in the PML body, a specific structure found inthe nucleus, and it binds to topoisomerase III (see Non-Patent Document18). The helicase has the unwinding activity of the G-quadruplexstructure, and thus is considered to contribute to telomere maintenance(see Non-Patent Document 19). Furthermore, the helicase has beenreported to unwind the Holliday junction and to interact with the Rad51protein (see Non-Patent Document 20). These findings suggest that BLMhelicase cooperates with other DNA-metabolizing enzymes and plays animportant role in recombinational DNA repair and telomere maintenance.

Of the five human proteins belonging to the RecQ DNA helicase family(RecQ1, BLM, WRN, RTS, and RecQ5), RecQ1, BLM, WRN, and RTS areexpressed at negligible levels in resting cells, but are expressed athigh levels in cells whose proliferation has been enhanced bytransformation with viruses (see Non-Patent Document 21). Furthermore,when the carcinogenic promoter TPA is added to resting cells, theexpression of RecQ1, BLM, WRN, and RTS is induced along with theinduction of cell division (see Non-Patent Document 21). These findingssuggest the importance of the RecQ DNA helicase family in cellproliferation.

Taken collectively, these findings suggest that the RecQ DNA helicasefamily members may be potential target molecules for anti-cancer therapybecause the family members participate in genomic repair in cells (BLM,WRN and RTS) and also in the maintenance of telomere structure (BLM andWRN), that they play important roles in the immortalization of certaincells (WRN), and that their expression is induced following celldivision (RecQ1, BLM, WRN and RTS).

However, even if a compound can suppress the proliferation of cancercells, if it has similar proliferation-suppressing effects on normalcells, that compound cannot be expected to be a useful anticancer agent.So far, nothing is known concerning how compounds that suppressexpression of RecQ1 genes act on normal cells, or whether such compoundshave cancer cell-specific cell proliferation-suppressing effects.

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DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide cancer cell-specificcell proliferation inhibitors aimed at suppressing expression of RecQ1helicase genes.

Means to Solve the Problems

The expression level of the RecQ DNA helicase family was found to besignificantly high in tumor cells and methods of screening for compoundsthat suppress tumor growth using the suppression of expression of RecQDNA helicase family genes as an index are known. It has also beensuggested that compounds suppressing RecQ helicase gene expression maysuppress cancer cell growth (see Japanese Patent Application KokaiPublication No. (JP-A) 2000-166600 (unexamined, published Japanesepatent application)).

However, the relationship between suppression of RecQ1 gene expressionand cancer cell-specific cell proliferation suppression has until nowbeen unknown.

Even if a certain compound is found to have cancer cellproliferation-suppressing effects, if it is unclear whether the compoundhas a proliferation-suppressing effect on normal cells, that compoundwould not be an effective pharmaceutical. This is because when such acompound also shows a proliferation-suppressing effect on normal cells,it carries the risk of side effects. In fact, to date, findingsindicating that various anticancer agents have side effects have beenreported (for example, Komarov P. G. et al., Science Vol. 285,1733-1737, 1999; Kamarova E. A. and Gudkov A. V. Biochemistry (Moscow)Vol. 65, 41-48, 2000; Botchkarev V. A. Cancer Research Vol. 60,5002-5006, 2000). If it is possible to develop pharmaceutical agentsthat have cancer cell-specific cell proliferation-suppressing effectsand do not act on normal cells, these agents will be expected to be veryuseful anticancer agents with few side effects.

The present inventors carried out dedicated research to achieve theabove-mentioned objectives. The expression of genes from the RecQ DNAhelicase family is known to be increased in tumor cell systems (forexample, cancer cells). The present inventors used siRNAs that exhibitthe effect of suppressing expression of the RecQ1 gene, which belongs tothe human RecQ helicase family genes, to examine the effect ofsuppressing RecQ1 gene expression on cancer cell proliferation. As aresult, the present inventors discovered that, although suppressing theexpression of the RecQ1 gene leads to observation of cellproliferation-suppressing effects in cancer cells, such effects are notseen in human TIG3 cells (normal diploid fibroblast cell line), whichare normal cells. Hence, the present inventors discovered for the firsttime that a cancer cell-specific cell proliferation-suppressing effectis observed as a result of suppressing RecQ1 gene expression. Therefore,the RecQ1 gene may be a target molecule for excellent carcinostaticagents with few side effects. Furthermore, the present inventorssucceeded in finding siRNA molecules with cancer cell-specific cellproliferation-suppressing effects. Pharmaceutical agents comprising suchmolecules are expected to be effective pharmaceuticals for treatingcancers with few side effects.

As described above, many of the existing anticancer agents have sideeffects; therefore, it would be very difficult to predict in advancethat a molecule having the effect of suppressing cancer cellproliferation will not act on normal cells, similarly to the siRNAmolecules of the present application against the RecQ1 gene. Therefore,the siRNA molecules provided by the present invention have advantageouseffects (cell proliferation-suppressing effects that are specific tocancer cells and do not affect normal cells) that cannot be predictedeven by those skilled in the art.

Thus, the present invention relates to cancer cell-specific cellproliferation inhibitors that target RecQ1 helicase gene expression, andparticularly relates to cancer cell-specific cell proliferationinhibitors comprising siRNAs with the effect of suppressing RecQ1 geneexpression. More specifically, the present invention provides thefollowing:

[1] a double-stranded RNA that can suppress the expression of an RecQ1gene by an RNAi effect, wherein the RNA comprises a structure in whichan RNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to32 or SEQ ID NOs: 40 to 43 and an RNA comprising a sequencecomplementary to said RNA are hybridized;[2] the double-stranded RNA of [1], which comprises a structure in whichone or more DNAs or RNAs overhang at an end;[3] a DNA vector that can express an RNA comprising the nucleotidesequence of any one of SEQ ID NOs: 1 to 32 or SEQ ID NOs: 40 to 43;[4] a cancer cell-specific cell proliferation inhibitor which comprisesthe RNA of [1] or [2], or the DNA of [3]; and[5] an anticancer agent comprising the cancer cell-specific cellproliferation inhibitor of [4] as an active ingredient. Theabove-mentioned cancer cells preferably refer to human cancer cells(cancer cells of human origin).

Furthermore, the present invention relates to:

[6] a method for suppressing cell proliferation cancer cell-specifically(a method for treating a cancer), which comprises the step ofadministering the RNA of [1] or [2] or the DNA of [3] to an individual(a subject, test subject, patient, etc.); and

[7] a use of the RNA of [1] or [2] or the DNA of [3] in the productionof a cancer cell-specific anticancer agent (a cancer cell-specific cellproliferation inhibitor).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences of the siRNAs against RecQ1 genethat were used in the Examples. All of the sequences are RNAs, and theoverhang sequence of all of the siRNAs is the deoxynucleotides ‘TT’.

FIG. 2 shows the expression levels of the RecQ1 gene in HeLa cells intowhich siRNAs against the RecQ1 gene have been introduced.

FIG. 3 shows the survival rates of HeLa cells 96 hours after siRNAsagainst the RecQ1 gene have been introduced.

FIG. 4 is a graph showing results of introducing siRNAs against theRecQ1 gene into TIG3 cells, and then quantifying the expression of mRNAs48 hours later by semi-quantitative RT-PCR. NS is a control siRNA. 15and 24 are the SEQ ID NOs of siRNAs shown in FIG. 1. The gene expressionobtained when a non-silencing siRNA was introduced was taken as 100%.

FIG. 5 is a graph indicating the survival rate of TIG3 cells 96 hoursafter introduction of siRNAs against the RecQ1 gene. NS is a controlsiRNA. 15 and 24 are the SEQ ID NOs of siRNAs shown in FIG. 1. The graphshows the number of cells when the number of cells after introduction ofa non-silencing siRNA was taken as 100%.

FIG. 6 shows the evaluation results of the medicinal effect of thesiRNAs against the RecQ1 gene. NT refers to untreated cancer-bearingmice.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors discovered that, by suppressing the expression ofthe RecQ1 gene, which belongs to the RecQ DNA helicase family genes,cell proliferation is suppressed cancer cell (tumor cell)-specifically.Further, the present inventors discovered RNA molecules that exhibiteffective cancer cell-specific cell proliferation-suppressing effectsthrough the suppression of RecQ1 gene expression by RNAi effects.

Therefore, firstly, the present invention provides RNAs (siRNAs andshRNAs) that can suppress RecQ1 gene expression by RNAi effects. SuchRNAs have cancer cell-specific cell proliferation-suppressing effects.In the present invention, the term “cancer cell-specific” refers toaction against cancer cells but substantial inaction (not showingeffective action) against normal cells. Cases in which the effectagainst normal cells is significantly less than the effect againstcancer cells are also comprised in the term “cancer cell-specific” ofthe present invention.

Those skilled in the art can readily obtain information on thenucleotide sequences of the RecQ1 genes of the present invention frompublic gene databases (for example, GenBank). Exemplary GenBankaccession numbers of the genes described above are listed below:

RecQ1 gene: NM_(—)002907 (SEQ ID NO: 33), NM_(—)032941 (SEQ ID NO: 34),BC001052 (SEQ ID NO: 35), D37984 (SEQ ID NO: 36), and L36140 (SEQ ID NO:37).

An example of an amino acid sequence of a protein encoded by a RecQ1gene of the present invention is indicated in SEQ ID NO: 38.

The RecQ1 genes of the present invention typically include, but are notlimited to, those derived from animals, more preferably those derivedfrom mammals, and most preferably those derived from humans.

The RNAs of the present invention that can suppress the expression ofRecQ1 genes by RNAi (RNA interference) effects (may be simply referredto as “the siRNAs of the present invention” in this application) aremore specifically, for example, RNAs comprising the nucleotide sequenceof any one of SEQ ID NOs: 1 to 32. Furthermore, examples of preferredembodiments of the siRNAs of the present invention includedouble-stranded RNAs (siRNAs) that include RNAs comprising thenucleotide sequence of any one of SEQ ID NOs: 1 to 32 as one of thestrands.

The present invention provides double-stranded RNAs which are RNAs(siRNAs) that can suppress RecQ1 gene expression by RNAi effects, andwhich comprise structures in which an RNA comprising the nucleotidesequence of any one of SEQ ID NOs: 1 to 32 and an RNA comprising asequence complementary to this RNA are hybridized.

For example, the siRNAs of the present invention that comprise thenucleotide sequence of SEQ ID NO: 1 (5′-cuacggcuuuggagauaua-3′) may beRNA molecules structured as below:

(herein, “I” indicates a hydrogen bond).

The above-mentioned RNA molecules that are structured such that one endis closed, for example, siRNAs comprising a hairpin structure (shRNAs),are also included in the present invention. Hence, molecules that canform an intramolecular double-stranded RNA structure are also comprisedin the present invention.

For example, molecules such as 5′-cuacggcuuuggagauaua-3′ (SEQ ID NO: 1)(xxxx)n uauaucuccaaagccguag (SEQ ID NO: 39)-3′ are also comprised in thepresent invention. (The aforementioned “(xxxx)n” indicates apolynucleotide comprising any nucleotide and any number of sequences.)

Preferred embodiments of the siRNAs of the present invention arepreferably double-stranded RNAs which are RNAs (siRNAs) that cansuppress RecQ1 gene expression by RNAi effects, and which comprise astructure in which an RNA comprising the nucleotide sequence of any oneof SEQ ID NOs: 1 to 32 and an RNA comprising a sequence complementary tothis RNA are hybridized. Double-stranded RNAs structured such that, forexample, there are one or more RNA additions or deletions at the end ofsuch a double-stranded RNA are also comprised in the present invention.In such cases, the RNAs forming the double strand are preferablyhomologous to a partial sequence of a RecQ1 gene. The length of theregion of the RNA forming a double strand in an siRNA of the presentinvention is ordinarily 15 to 30 bp, preferably 15 to 27 bp or so, morepreferably 19 to 21 bp, and most preferably 19 bp (for example, an siRNAin which one of the strands is an RNA of any one of SEQ ID NOs: 1 to32), but the length is not necessarily limited thereto.

All of the nucleotides in the siRNAs of the present invention are notnecessarily required to be ribonucleotides (RNAs). Namely, in thepresent invention, one or more of the ribonucleotides composing thesiRNAs may be corresponding deoxyribonucleotides. “Corresponding” meansthat the nucleotides have identical base species (adenine, guanine,cytosine, and thymine (uracil)), but that the structure of the sugarportion is different. For example, the deoxyribonucleotide correspondingto a ribonucleotide with adenine means a deoxyribonucleotide withadenine. In addition, the above “more” is not limited to a particularnumber but preferably means a small number around two to five.

In general, the term “RNAi” refers to a phenomenon where target geneexpression is inhibited by inducing disruption of the target gene mRNAs.This disruption is caused by introducing into cells a double-strandedRNA that comprises, a) a sense RNA comprising a sequence homologous to atarget gene mRNA sequence, and b) an antisense RNA comprising a sequencecomplementary to the sense RNA. While the precise RNAi mechanism remainsunclear, it is thought that an enzyme called DICER (a member of theRNase III nuclease family) contacts the double-stranded RNA, degradingit into small fragments called “small interfering RNAs” or “siRNAs”. Thedouble-stranded RNAs of the present invention comprising the RNAieffects preferably refer to these siRNAs.

In a preferred embodiment of the present invention, the double-strandedRNAs are RNAs that can suppress RecQ1 gene expression by RNAi effectsand that comprise a structure in which an RNA comprising the nucleotidesequence of any one of SEQ ID NOs: 1 to 32 and an RNA comprising asequence complementary to this RNA are hybridized.

Furthermore, DNAs that allow the expression of the siRNAs(double-stranded RNAs) of the present invention are also included in thepresent invention. Specifically, the present invention provides DNAs(vectors) that allow the expression of double-stranded RNAs of thepresent invention. These DNAs (vectors) that allow the expression ofdouble-stranded RNAs of the present invention are typically DNAscomprising a structure where a DNA encoding one strand of thedouble-stranded RNA and a DNA encoding the other strand of thedouble-stranded RNA are operably linked to a promoter. Those skilled inthe art can readily prepare an above-described DNA of the presentinvention with common genetic engineering techniques. More specifically,expression vectors of the present invention can be prepared byappropriately inserting DNAs encoding RNAs of the present invention intovarious known expression vectors.

Generally, the double-stranded RNAs having an RNAi effect aredouble-stranded RNAs comprising a sense RNA, which comprises a sequencehomologous to a continuous RNA region in the mRNA of a target gene whoseexpression is to be suppressed, and an antisense RNA, which comprises asequence complementary to the sense RNA.

In general, since double-stranded RNAs with an overhang of severalnucleotides on one end have strong RNAi effects, the double-strandedRNAs of the present invention preferably comprise an overhang of severalnucleotides on an end. The length of the nucleotides forming theoverhang as well as the sequence are not particularly limited. Thisoverhang may be DNA or RNA. For example, the overhang preferably has twonucleotides. A double-stranded RNA comprising an overhang of, forexample, TT (a thymine doublet), UU (a uracil doublet), or some othernucleotide (most preferably, a molecule comprising a double-stranded RNAof 19 nucleotides and an overhang of two nucleotides (TT)) can besuitably used in the present invention. The double-stranded RNAs of thepresent invention also include molecules in which the overhangingnucleotides are DNAs.

Examples of the siRNA molecules of the present invention where thenucleotides of the overhang portion are TT include molecules having TTadded to their 3′ side, such as the molecule indicated below:

The above-mentioned “double-stranded RNAs having an RNAi effect on RecQ1genes” of the present invention can be suitably produced by thoseskilled in the art based on the nucleotide sequences disclosed in thepresent description. Specifically, the double-stranded RNAs of thepresent invention can be produced based on the nucleotide sequence ofany one of SEQ ID NOs: 1 to 32. If one of the strands has beendetermined (for example, a nucleotide sequence described in any one ofSEQ ID NOs: 1 to 32), the nucleotide sequence of the other strand (thecomplementary strand) can be easily determined by those skilled in theart. siRNAs of the present invention can be suitably produced by thoseskilled in the art using commercially available nucleic acidsynthesizers. Common custom synthesis services can also be used tosynthesize desired RNAs.

Since the siRNAs of the present invention (for example, adouble-stranded RNA molecule in which one of the strands has thenucleotide sequence of any one of SEQ ID NOs: 1 to 32) have cancercell-specific cell proliferation-suppressing effects, the presentinvention provides cancer cell-specific cell proliferation inhibitorsthat comprise an siRNA of the present invention as an active ingredient.

If the cancer cell proliferation-suppressing effect in the presentinvention arises from the induction of apoptosis, the siRNAs of thepresent invention will be expected to be cancer cell-specificapoptosis-inducing agents.

The term “apoptosis” generally refers to cell death actively induced bythe cell itself under physiological condition. The morphologicalfeatures of apoptosis include, for example, chromosome condensation inthe cell nucleus, nuclear fragmentation, loss of microvilli on the cellsurface, and cytoplasmic shrinkage. Thus, as used herein, the term“apoptosis-inducing effect” refers to, for example, the effect ofinducing in cells the above-described morphological features ofapoptosis, but is not limited to those described above. One skilled inthe art can appropriately assess whether or not apoptosis is beinginduced in cells.

For example, the present invention's apoptosis inducers specific forcancer cells are expected to be anticancer agents (carcinostatic agents)having apoptosis-inducing activity as their mechanism of action.

The present invention provides anticancer agents (pharmaceuticalcompositions for cancer therapy) that comprise a cancer cell-specificcell proliferation inhibitor of the present invention as an activeingredient.

Pharmaceutical agents of the present invention can be provided as amixture with a pharmaceutically acceptable carrier. Suchpharmaceutically acceptable carriers can include, but are not limitedto, for example, detergents, excipients, coloring agents, flavoringagents, preservatives, stabilizers, buffers, suspensions, isotonizingagents, binders, disintegrating agents, lubricants, fluidizing agents,and correctives. Other conventional carriers can be also usedappropriately.

The pharmaceutical agents of the present invention can be formulated byadding the above-indicated carriers as required and according toconventional methods. Specifically, such carriers include: lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinylacetaldiethylamino acetate,polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethylcellulose, cornstarch, and inorganic salts.

The dosage forms for the agents described above include, for example,oral forms, such as tablets, powders, pills, dispersing agents,granules, fine granules, soft and hard capsules, film-coated tablets,pellets, sublingual tablets, and pastes; and parenteral forms, such asinjections, suppositories, endermic liniments, ointments, plasters, andliquids for external use. Those skilled in the art can select theoptimal dosage form depending on the administration route, subject, andsuch.

Viral vectors such as retroviruses, adenoviruses, and Sendai viruses andnon-viral vectors such as liposomes can be used to administer DNAsexpressing the siRNAs of the present invention that suppress the RecQ1genes into living bodies. Alternatively, non-viral vectors such asliposomes, polymer micelles, or cationic carriers, may be used toadminister synthetic siRNAs of the present invention that suppress theRecQ1 genes into living bodies. The administration methods include, forexample, in-vivo and ex-vivo methods.

The present invention also comprises the above-described pharmaceuticalcompositions having cancer cell-specific cell proliferation-suppressingeffect. Ultimately, the doses of the pharmaceutical agents orpharmaceutical compositions of the present invention can beappropriately determined by a physician considering the type of dosageform, administration method, patient's age, weight, symptoms, and so on.

The types of cancers for which a cell proliferation-suppressing effectis expected in the present invention are not particularly limited, butexamples include breast cancers, lung cancers, osteosarcomas, cervicalcancers, fibrosarcomas, ovarian teratocarcinomas, embryonal cancers,bladder cancers, chronic myeloid leukemias, acute lymphoblasticleukemias, glioblastomas, liver cancers, glioblastomas, melanomas,kidney cancers, pancreatic cancers, stomach cancers, prostate cancers,and such.

Furthermore, the present invention relates to methods for suppressingcancer cell-specifically (cancer cell-specific methods for treatingcancer) and methods for suppressing cell proliferation cancercell-specifically, which comprise the step of administering an RNA orDNA of the present invention or a pharmaceutical agent of the presentinvention to individuals (for example, patients) or to cellular tissues(cancer cell tissues and such).

The individuals in the methods of the present invention are preferablyhumans, but are not particularly limited thereto, and they may benon-human animals.

In general, administration to individuals can be carried out by methodsknown to those skilled in the art, examples of which includeintra-arterial injection, intravenous injection, and subcutaneousinjection. Although the dosage varies depending on the weight and age ofthe subject (patient and such), the administration method, and so on,suitable dosages can be appropriately selected by those skilled in theart.

Moreover, the present invention relates to the uses of the RNAs or DNAsof the present invention, or to uses of the pharmaceutical agents of thepresent invention, in the production of cancer cell-specific cellproliferation inhibitors or anticancer agents.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

The present invention will be described in detail below with referenceto Examples, but is not to be construed as being limited thereto.

Example 1 Cell Cultures

HeLa cells (human cervical cancer cells) were used as human cancercells, and TIG3 cells (normal diploid fibroblast cells) were used asnormal human cells. HeLa cells and TIG3 cells were cultured at 37° C.under 5% CO₂ using Dulbecco's modified Eagle's medium containing 10%fetal bovine serum and 50 μg/mL gentamicin.

Example 2 siRNA Design

Thirty-two siRNAs against RecQ1 gene were designed according to themethod of Elbasher et al. (Elbasher, M. S. et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494-498 (2001)) and the method of Reynolds et al. (ReynoldsA. et al., Rational siRNA design for RNA-interference. Nat. Biotechnol.3, 326-30 (2004)). FIG. 1 shows each of the siRNA sequences. The siRNAswere synthesized at Qiagen.

Example 3 Cancer Cell-Specific Cell Proliferation-Suppressing EffectsDue to the Suppression of RecQ1 Gene Expression

(1) Suppression of RecQ1 Gene Expression by siRNAs

Cells were plated onto 24-well plates at a density of 0.8-1.5×10⁴cells/well 24 hours before transfection, and siRNAs were transfectedunder the condition of 20-50% confluency. 10 pmol of siRNA wastransfected per well using Oligofectamine (Invitrogen) or Lipofectamine2000 (Invitrogen) following the manufacturer's protocol. Expression ofthe RecQ1 gene mRNA 24 hours after introduction of siRNA was quantifiedusing Taqman PCR. Specifically, total RNA was extracted from cells at 24hours after siRNA transfection using an RNeasy Mini Kit (Qiagen). ABIPRISM 7000 Sequence Detection System (Applied Biosystems) was used forquantitative PCR. RT-PCR primers for the RecQ1 gene and β-actin gene,and TaqMan probes were purchased from Applied Biosystems. RT-PCRreactions were performed using a QuantiTect Probe RT-PCR Kit (Qiagen)according to the manual. Expression of RecQ1 mRNA was quantitativelycompared using β-actin as a standard. The expression level of the RecQ1gene mRNA in cells into which control siRNAs that do not affect RecQ1gene expression had been transfected was defined as 100%, and the RecQ1mRNA expressions in cells into which each siRNA had been introduced werecompared.

(2) Cell Proliferation Assays

siRNA transfection was performed under the same conditions as describedabove, and 96 hours later, viable cells were measured using a viablecell count reagent SF (Nakalai Tesque). The experiment was carried outat N=3, and average values were calculated. The viable cell count ofcells into which control siRNA that does not affect RecQ1 geneexpression was introduced was defined as 100%, and the viable cellcounts for cells into which each siRNA was introduced were calculated.

(3) Results

Using HeLa cells, which are human cervical cancer cells, the effects oncell proliferation of suppression of RecQ1 gene expression by siRNAswere investigated. As a result of individually transfecting the 32 typesof siRNAs against the RecQ1 gene into HeLa cells, a geneexpression-suppressing effect of 70% or more was observed for all of thesiRNAs (FIG. 2). Under such conditions, when the number of viable HeLacells after 96 hours was compared to that of the NS-siRNA-treated group,a proliferation suppression of 30% or more was observed in all of thesiRNA-treated groups (FIG. 3).

Next, the effects on the proliferation of normal cells were investigatedusing TIG3 cells. When the siRNAs of SEQ ID NOs: 15 and 24, which showedstrong proliferation-suppressing effects in HeLa cells, wereindividually introduced into TIG3 cells, each of them suppressed RecQ1gene expression by approximately 70% (FIG. 4). Under such conditions,when the number of viable TIG3 cells after 96 hours was compared to thatof the NS-siRNA-treated group, no effect on the proliferation of TIG3cells was recognized (FIG. 5).

These results proved that RecQ1-siRNA strongly inhibits theproliferation of cancer cells, but hardly affects the proliferation ofnormal cells.

Example 4 Proliferation Inhibition of Tumor Cells by siRNAs inCancer-Bearing Animal Models

The sequences of the siRNAs and 27 mer dsRNA used in the animal studiesare shown below:

TABLE 1 Sequences of siRNAs against RecQ1 used in animal studies siRNAsequence 24 GGGCAAUCAGGAAUCAUAU (SEQ ID NO: 24) 33 GCUUGAAACUAUUAACGUA(SEQ ID NO: 40) 34 UAAGACCACAGUUCAUAGA (SEQ ID NO: 41) 35GUUAUCCAUCAUUCAAUGA (SEQ ID NO: 42)All siRNA sequences are RNAs.The overhang sequence of all of the siRNAs is the deoxynucleotides ‘TT’.The 27 mer dsRNA sequence against RecQ1 used in animal studies

36 GGAAAAGUUCAGACCACUUCAGCUUGA (SEQ ID NO: 43)The dsRNA sequence is all RNA and does not have an overhang.

The RecQ1 gene expression levels in HeLa cells treated with the aboveRecQ1-siRNAs are the following:

TABLE 2 Gene expression level 33 3% 34 19% 35 18% 36 6% NS 100%

The present inventors also examined whether proliferation inhibition oftumor cells by siRNAs against RecQ1 helicase will also occur incancer-bearing animal models. The siRNAs and 27 mer dsRNA against theRecQ1 gene shown above were used.

Male BALB/cA nude mice were purchased from CLEA Japan, Inc. A549 cells(5×10⁶ cells/0.1 mL) were subcutaneously transplanted into the back ofnude mice (seven weeks old). siRNA administration began on the eighthday after tumor cell transplantation. With regard to RecQ1-siRNA, 22 μgof siRNA with phosphorylated 5′ end was mixed with 5 μg ofpolyethylenimine (molecular weight of 10,000, Wako) in 50 μL ofphysiological saline. This mixture was subcutaneously injected sixtimes, once every three days (on days 8, 11, 14, 17, 20, and 23), intothe uppermost part of the solid tumor. The tumor volume was measuredusing calipers. The equation for calculating tumor volume was L×W²/2.Herein, L is the major axis and W is the minor axis of the tumor.Statistical significance of the tumor volume was analyzed using t-tests.

As a result, all RecQ1-siRNAs suppressed tumor growth, but NS-siRNA (ansiRNA which does not affect the expression of human and mouse genes),which was similarly mixed with polyethylenimine, had no effect and tumorvolume increased (FIG. 6). Mice administered with a mixture ofRecQ1-siRNA and polyethylenimine did not show a reduction in weightcompared to non-cancer-bearing mice, which indicated that this treatmentdoes not have serious side effects.

The studies by the present inventors revealed that silencing of RecQ1helicase expression causes suppression of tumors in cancer-bearinganimal models.

INDUSTRIAL APPLICABILITY

Even if a certain compound is found to have the effect of suppressingcancer cell proliferation, use of that compound as a pharmaceutical isdifficult when it is unclear whether it also has the effect ofsuppressing the proliferation of normal cells. This is because when sucha compound also shows cell proliferation-suppressing effect on normalcells, it carries with it the risk of side effects. Hence, if the cellproliferation-suppressing effect is not cancer cell-specific, it wouldordinarily be difficult to actually use the compound as apharmaceutical. The pharmaceutical agents of the present invention(nucleic acids having RNAi effects) can be said to be very practical andhighly effective pharmaceutical agents, since their cellproliferation-suppressing effect is cancer cell-specific.

1. A double-stranded RNA that can suppress the expression of an RecQ1gene by an RNAi effect, wherein the RNA comprises a structure in whichan RNA comprising the nucleotide sequence of SEQ ID NO: 41 and an RNAcomprising a sequence complementary to said RNA are hybridized.
 2. Thedouble-stranded RNA of claim 1, which comprises a structure in which oneor more DNAs or RNAs overhang at an end.
 3. A DNA vector that canexpress an RNA comprising the nucleotide sequence of SEQ ID NO:
 41. 4. Acancer cell-specific cell proliferation inhibitor which comprises theRNA of claim 1 or 2, or the DNA of claim
 3. 5. An anticancer agentcomprising the cancer cell-specific cell proliferation inhibitor ofclaim 4 as an active ingredient.